1. Field of the Invention
This invention relates to a method of spotting sample liquid such as blood or urine with a spotting tip onto a frameless dry (dry-to-the-touch) chemical analysis film having a reagent layer containing therein a reagent whose optical density changes by chemical reaction, biochemical reaction, immunoreaction or the like with a specific biochemical component (the object of measurement; sometimes called "analyte" or "item of object") contained in the sample liquid. More particularly, this invention relates to such a method which, when a sample liquid is to be spotted onto a plurality of frameless dry chemical analysis films for different analytes in sequence, can prevent deterioration in accuracy of measurement for frameless dry chemical analysis films which are spotted with the sample liquid later due to contamination of the spotting tip by a substance on the chemical analysis films which are spotted earlier.
2. Description of the Related Art
There has been put into practice a dry chemical analysis film (sometimes referred to as "multi-layered chemical analysis element") with which a specific biochemical component or the like contained in a sample liquid can be quantitatively analyzed through a droplet of the sample liquid spotted onto the chemical analysis film. When a biochemical component or the like contained in a sample liquid is quantitatively analyzed using such a chemical analysis film, a droplet of the sample liquid is spotted onto the chemical analysis film and is held at a constant temperature for a predetermined time (incubation) in an incubator to cause coloring reaction. Then measuring light containing a wavelength which is pre-selected according to the combination of the component to be analyzed and the reagent contained in the reagent layer of the chemical analysis film is projected onto the chemical analysis film and the reflective optical density of the chemical analysis film is measured. The concentration in the sample liquid of the component to be analyzed is determined on the basis of the reflective optical density using a calibration curve or a standard curve which represents the relation between the concentration of the component and the reflective optical density.
The dry chemical analysis film comprises a support sheet such as of a plastic sheet and a reagent layer which contains a reagent and is formed on the support sheet. Conventionally the chemical analysis film is generally used in the form of a chemical analysis slide comprising a chip of the chemical analysis film held by an organic polymer or a plastic frame.
However use of such chemical analysis films with frame results in increase in the sizes of various parts handling the films such as cells in the incubator for incubating the films, the transfer system, a film supplier for storing the films in dry state, a chemical analysis film cartridge and the like. Thus the frame of the chemical analysis slide is obstructive to reducing the size of the biochemical analysis apparatus and at the same time reduces the film accommodating capacity of the incubator and the film supplier, which obstructs increase in the overall throughput capacity of the biochemical analysis apparatus. Further cost for mounting the frame is high and adds to cost of biochemical analysis.
In view of these circumstances, we have previously proposed a system of biochemical analysis in which a chip of chemical analysis film as it is or without film is used. The chemical analysis film chip without frame will be referred to as "frameless chemical analysis film" in order to distinguish it from the chemical analysis slide, i.e., the chemical analysis film chip with frame, hereinbelow.
In the case of a chemical analysis slide, the sample liquid is spotted in the following manner. That is, after a droplet of the sample liquid is formed at the end of the spotting tip, the spotting tip is lowered toward the surface of the slide and is stopped when the droplet is brought into contact with the chemical analysis film of the slide, thereby causing the droplet to spread over the surface of the film.
In the method described in U.S. Pat. No. 4,340,390, the lower end of the spotting tip is stopped at a predetermined distance from the surface of the film and then the sample liquid in the spotting tip is discharged at a predetermined rate.
However when such methods of spotting the sample liquid on the chemical analysis slide are used for the frameless chemical analysis film, the following difficulties are encountered. That is, though in the case of chemical analysis slides in which the surface of the film is kept flat by the frame, the position of the film surface can be held constant irrespective of the kind of the chemical analysis film, the position of the film surfaces is apt to vary according to the kind of the chemical analysis film in the case of the frameless chemical analysis film since the frameless chemical analysis film is warped or curled or curved in a dry state and the degree of warp, curl or curve varies depending on the kind of the chemical analysis film.
Accordingly when the distance between the end of the spotting tip and the film surface is too large, the droplet or the discharged flow of the sample liquid is apt to break near the end of the spotting tip and part of the sample liquid is apt to remain in the spotting tip. This phenomenon is more apt to occur when the sample liquid spreads over the chemical analysis film at a high rate and when the viscosity of the sample liquid is low.
On the other hand, when the distance between the end of the spotting tip and the film surface is too small, the sample liquid stands up above the film surface and the end portion of the spotting tip dips into the sample liquid on the film surface. As a result, part of the sample liquid remains adhering to the outer surface of the end portion of the spotting tip after end of spread of the sample liquid. This phenomenon is more apt to occur when the sample liquid spreads over the chemical analysis film at a low rate and when the viscosity of the sample liquid is high.
Further in the spotting method described in U.S. Pat. No. 5,143,849, a meniscuscoid droplet is formed on the end of the spotting tip and the spotting tip is lowered toward the film. The spotting tip is stopped when that the droplet is brought into contact with the film surface is detected through a fine change in the pressure in the spotting tip, and the sample liquid in the spotting tip is discharged in this state or after the spotting tip is lifted by a predetermined amount.
With this method, the distance between the end of the spotting tip and the film surface can be precisely controlled and accordingly the problem inherent to the two prior arts described above can be avoided. However since the meniscuscoid droplet is small in size, it takes a long time to detect whether the droplet is in contact with the film surface and accordingly it is difficult to lower the spotting tip at a high speed, which makes it difficult to shorten the spotting time.
There are plurality of analytes for a sample liquid such as blood, urine or the like, and different chemical analysis films are used for the respective analytes.
When a sample liquid is spotted onto a plurality of different frameless chemical analysis films in sequence, there is a fear that accuracy of measurement can deteriorate depending on the order in which the frameless chemical analysis films are spotted with the sample liquid. That is, since it is difficult to keep constant the position of the surface of the frameless chemical analysis film, i.e., the level of a spreading layer or a blood cell filtering layer on which the sample liquid is spotted, for the reason described above, it has been proposed a method of spotting a sample liquid in which the end of the spotting tip is once brought into contact with the surface of the chemical analysis film and then the sample liquid is discharged from the spotting tip while moving upward the spotting tip in order to make constant the distance between the end of the spotting tip and the film surface, which greatly affects the accuracy of measurement. (European Unexamined Patent Publication No. 0677744)
When the spotting tip is brought into contact with the surface of the chemical analysis film, one or more of the components of the reagent layer of the chemical analysis film can adhere to the spotting tip, and since a spotting tip is used when a sample liquid is spotted onto a plurality of chemical analysis films, the component of the reagent layer of the preceding chemical analysis film adhering to the spotting tip can be transferred to the reagent layer of the next chemical analysis film through the contact between the spotting tip and the reagent layer or can be dissolved into the sample liquid and spotted onto the next chemical analysis film together with the sample liquid. When the reagent layer of the next chemical analysis film is thus contaminated, the measured concentration or activity of the specific analyte to be analyzed with the chemical analysis film can be affected by the contaminant, which can lead to a large measuring error.
For example, when the analyte for a frameless chemical analysis film is LDH (lactate dehydrogenase) and the analyte for the next frameless chemical analysis film is GPT (glutamic-pyruvic transaminase), GPT is contained in the reagent layer of the chemical analysis film for LDH and part of the GPT component can adhere to the spotting tip when the spotting tip is brought into contact with the chemical analysis film for LDH. When the GPT component adhering to the spotting tip is transferred to the chemical analysis film for GPT, the GPT component adds to the inherent GPT content (or activity) of the sample liquid, whereby the measured value of the GPT content becomes larger than the real value.
Actually, our experiment has revealed that the measured value (activity) of the GPT becomes larger than the actual value by 1.5 to 2 U/L (U represents international unit and L represents liter) when the sample liquid is spotted onto the frameless chemical analysis film for GPT with the spotting tip in contact with the film after the frameless chemical analysis film for LDH is spotted with the sample liquid with the spotting tip in contact with the film as will be described later with reference to FIGS. 7A and 7B. Since the measured value of GPT normally should be in the range of 4 to 36 U/L, increase of the measured value over the actual value by 1.5 to 2 U/L can lead to misdiagnosis that the sample liquid whose actual GPT value is 34 to 36 U/L is diseased (disease in heart, liver or bile biliary duct or malignant adenoma).